Plasmodium falciparum glyoxalase II: Theorell-Chance product inhibition patterns, rate-limiting substrate binding via Arg(257)/Lys(260), and unmasking of acid-base catalysis

Glyoxalase II (GloII) is a ubiquitous thioester hydrolase catalyzing the last step of the glutathione-dependent conversion of 2-oxoaldehydes to 2-hydroxycarboxylic acids. Here, we present a detailed structure-function analysis of cGloII from the malaria parasite Plasmodium falciparum. The activity of the enzyme was salt-sensitive and pH-log k(cat) and pH-log k(cat)/K-m profiles revealed acid-base catalysis. An acidic pK(a)(app) value of approximately 6 probably reflects hydroxide formation at the metal center. The glutathione-binding site was analyzed by site-directed mutagenesis. Substitution of residue Arg(154) caused a 2.5-fold increase of K-m(app), whereas replacements of Arg(257) or Lys(260) were far more detrimental. Although the glutathione-binding site and the catalytic center are separated, six of six single mutations at the substrate-binding site decreased the k(cat)(app) value. Furthermore, product inhibition studies support a Theorell-Chance Bi Bi mechanism with glutathione as the second product. We conclude that the substrate is predominantly bound via ionic interactions with the conserved residues Arg(257) and Lys(260), and that correct substrate binding is a pH-and salt-dependent rate-limiting step for catalysis. The presented mechanistic model is presumably also valid for GloII from many other organisms. Our study could be valuable for drug development strategies and enhances the understanding of the chemistry of binuclear metallohydrolases

Relationship between cultivation mode of white rot fungi and their efficiency for olive oil mill wastewaters treatment

Four white rot fungi (WRF) strains, Phanerochaete chrysosporium , Trametes versicolor , Coriolopsis polyzona and Pycnoporus coccineus , were tested for efficiency of treatment of Olive Oil mill wastewaters (OOMW) in relation with their cultivation mode, i.e. under the form of free mycelium, mycelium immobilized in alginate beads and solid state cultivation on Petri dishes. Study of biodegradation of phenolic compounds, chemical oxygen demand (COD) decrease and decolourisation of OOMW have shown that Coriolopsis polyzona and Pycnoporus coccineus degradation performances were apparently only slightly affected by the cell cultivation procedures experienced here. In contrast, Phanerochaete chrysosporium and Trametes versicolor showed respectively marked preferences for solid state and alginate immobilisation procedures. Both mono and polyphenolics were reduced to different extent during incubation depending on the strain, as shown by gel filtration analysis. Final pH obtained after fungal treatment of the OOMW based medium (initial pH of 5.0) was measured in order to evaluate the possibility of releasing friendly the treated wastewater in the environment. Laboratory studies as reported here may be useful for orienting the choice of a strain for treating pollution by OOMW in a particular real situation